Harsh P. Bais

Root Exudation and Rhizosphere Biology

Our understanding of the biology, biochemistry, and genetic development of roots has considerably improved during the last decade ([Smith and Fedoroff, 1995][1]; [Flores et al., 1999][2];[Benfey and Scheres, 2000][3]). In contrast, the processes mediated by roots in the rhizosphere such as the

Biocontrol of Bacillus subtilis against Infection of Arabidopsis Roots by Pseudomonas syringae Is Facilitated by Biofilm Formation and Surfactin Production

Relatively little is known about the exact mechanisms used by Bacillus subtilis in its behavior as a biocontrol agent on plants. Here, we report the development of a sensitive plant infection model demonstrating that the bacterial pathogen Pseudomonas syringae pv tomato DC3000 is capable of infecting Arabidopsis roots both in vitro and in soil. Using this infection model, we demonstrated the biocontrol ability of a wild-type B. subtilis strain 6051 against P. syringae. Arabidopsis root surfaces treated with B. subtilis were analyzed with confocal scanning laser microscopy to reveal a three-dimensional B. subtilis biofilm. It is known that formation of biofilms by B. subtilis is a complex process that includes secretion of surfactin, a lipopeptide antimicrobial agent. To determine the role of surfactin in biocontrol by B. subtilis, we tested a mutant strain, M1, with a deletion in a surfactin synthase gene and, thus, deficient in surfactin production. B. subtilis M1 was ineffective as a biocontrol agent against P. syringae infectivity in Arabidopsis and also failed to form robust biofilms on either roots or inert surfaces. The antibacterial activity of surfactin against P. syringae was determined in both broth and agar cultures and also by live-dead staining methods. Although the minimum inhibitory concentrations determined were relatively high (25 μg mL-1), the levels of the lipopeptide in roots colonized by B. subtilis are likely to be sufficient to kill P. syringae. Our results collectively indicate that upon root colonization, B. subtilis 6051 forms a stable, extensive biofilm and secretes surfactin, which act together to protect plants against attack by pathogenic bacteria.

Root-Secreted Malic Acid Recruits Beneficial Soil Bacteria

Beneficial soil bacteria confer immunity against a wide range of foliar diseases by activating plant defenses, thereby reducing a plants susceptibility to pathogen attack. Although bacterial signals have been identified that activate these plant defenses, plant metabolites that elicit rhizobacterial responses have not been demonstrated. Here, we provide biochemical evidence that the tricarboxylic acid cycle intermediate l-malic acid (MA) secreted from roots of Arabidopsis (Arabidopsis thaliana) selectively signals and recruits the beneficial rhizobacterium Bacillus subtilis FB17 in a dose-dependent manner. Root secretions of l-MA are induced by the foliar pathogen Pseudomonas syringae pv tomato (Pst DC3000) and elevated levels of l-MA promote binding and biofilm formation of FB17 on Arabidopsis roots. The demonstration that roots selectively secrete l-MA and effectively signal beneficial rhizobacteria establishes a regulatory role of root metabolites in recruitment of beneficial microbes, as well as underscores the breadth and sophistication of plant-microbial interactions.

Enantiomeric-Dependent Phytotoxic and Antimicrobial Activity of (±)-Catechin. A Rhizosecreted Racemic Mixture from Spotted Knapweed

In this communication, we unravel part of the mystery surrounding the allelopathic capability of the noxious weed spotted knapweed ( Centaurea maculosa ). We have found that (−)-catechin is a root-secreted phytotoxin that undoubtedly contributes to spotted knapweeds invasive behavior in the

Pseudomonas aeruginosa-plant root interactions. Pathogenicity, biofilm formation, and root exudation

Pseudomonas aeruginosa is an opportunistic human pathogen capable of forming a biofilm under physiological conditions that contributes to its persistence despite long-term treatment with antibiotics. Here, we report that pathogenic P. aeruginosa strains PAO1 and PA14 are capable of infecting the roots of Arabidopsis and sweet basil (Ocimum basilicum), in vitro and in the soil, and are capable of causing plant mortality 7 d postinoculation. Before plant mortality, PAO1 and PA14 colonize the roots of Arabidopsis and sweet basil and form a biofilm as observed by scanning electron microscopy, phase contrast microscopy, and confocal scanning laser microscopy. Upon P. aeruginosa infection, sweet basil roots secrete rosmarinic acid (RA), a multifunctional caffeic acid ester that exhibits in vitro antibacterial activity against planktonic cells of both P. aeruginosa strains with a minimum inhibitory concentration of 3 μg mL-1. However, in our studies RA did not attain minimum inhibitory concentration levels in sweet basils root exudates before P. aeruginosa formed a biofilm that resisted the microbicidal effects of RA and ultimately caused plant mortality. We further demonstrated that P. aeruginosa biofilms were resistant to RA treatment under in vivo and in vitro conditions. In contrast, induction of RA secretion by sweet basil roots and exogenous supplementation of Arabidopsis root exudates with RA before infection conferred resistance to P. aeruginosa. Under the latter conditions, confocal scanning laser microscopy revealed large clusters of dead P. aeruginosa on the root surface of Arabidopsis and sweet basil, and biofilm formation was not observed. Studies with quorum-sensing mutants PAO210 (ΔrhlI), PAO214 (ΔlasI), and PAO216 (ΔlasI ΔrhlI) demonstrated that all of the strains were pathogenic to Arabidopsis, which does not naturally secrete RA as a root exudate. However, PAO214 was the only pathogenic strain toward sweet basil, and PAO214 biofilm appeared comparable with biofilms formed by wild-type strains of P. aeruginosa. Our results collectively suggest that upon root colonization, P. aeruginosa forms a biofilm that confers resistance against root-secreted antibiotics.

Proton-Transfer-Reaction Mass Spectrometry as a New Tool for Real Time Analysis of Root-Secreted Volatile Organic Compounds in Arabidopsis

Plant roots release about 5% to 20% of all photosynthetically-fixed carbon, and as a result create a carbon-rich environment for numerous rhizosphere organisms, including plant pathogens and symbiotic microbes. Although some characterization of root exudates has been achieved, especially of secondary metabolites and proteins, much less is known about volatile organic compounds (VOCs) released by roots. In this communication, we describe a novel approach to exploring these rhizosphere VOCs and their induction by biotic stresses. The VOC formation of Arabidopsis roots was analyzed using proton-transfer-reaction mass spectrometry (PTR-MS), a new technology that allows rapid and real time analysis of most biogenic VOCs without preconcentration or chromatography. Our studies revealed that the major VOCs released and identified by both PTR-MS and gas chromatography-mass spectrometry were either simple metabolites, ethanol, acetaldehyde, acetic acid, ethyl acetate, 2-butanone, 2,3,-butanedione, and acetone, or the monoterpene, 1,8-cineole. Some VOCs were found to be produced constitutively regardless of the treatment; other VOCs were induced specifically as a result of different compatible and noncompatible interactions between microbes and insects and Arabidopsis roots. Compatible interactions of Pseudomonas syringae DC3000 and Diuraphis noxia with Arabidopsis roots resulted in the rapid release of 1,8-cineole, a monoterpene that has not been previously reported in Arabidopsis. Mechanical injuries to Arabidopsis roots did not produce 1,8-cineole nor any C6 wound-VOCs; compatible interactions between Arabidopsis roots and Diuraphis noxia did not produce any wound compounds. This suggests that Arabidopsis roots respond to wounding differently from above-ground plant organs. Trials with incompatible interactions did not reveal a set of compounds that was significantly different compared to the noninfected roots. The PTR-MS method may open the way for functional root VOC analysis that will complement genomic investigations in Arabidopsis.

Curcumin, a known phenolic from Curcuma longa, attenuates the virulence of Pseudomonas aeruginosa PAO1 in whole plant and animal pathogenicity models.

The effect of curcumin on the virulence of Pseudomonas aeruginosa (PAO1) using whole plant and animal pathogenicity models was investigated. The effect of curcumin on PAO1 virulence was studied by employing in vitro assays for virulence factor production, Arabidopsis thaliana/Caenorhabditis elegans pathogenicity models, and whole genome microarray analysis. It is shown that the curcumin inhibits PAO1 virulence factors such as biofilm formation, pyocyanin biosynthesis, elastase/protease activity, and acyl homoserine lactone (HSL) production. As a consequence of this, curcumin treatment resulted in the reduced pathogenicity of P. aeruginosa-C. elegans and P. aeruginosa-A. thaliana infection models. In addition, transcriptome analysis of curcumin-treated PAO1 revealed down-regulation of 31 quorum sensing (QS) genes, of which many have already been reported for virulence. The supplementation of HSLs along with the curcumin treatment resulted in increased pathogencity and recovery of higher bacterial titers in a plant pathogenecity model. These data reveal the involvement of curcumin in QS interruption to reduce pathogenicity. Curcumin attenuates PAO1 virulence by down-regulation of virulence factors, QS, and biofilm initiation genes. The effect of curcumin on multiple targets such as virulence, QS, and biofilm initiation makes curcumin a potential supplemental molecule for the treatment of P. aeruginosa infections.

Mediation of pathogen resistance by exudation of antimicrobials from roots

Most plant species are resistant to most potential pathogens. It is not known why most plant–microbe interactions do not lead to disease, although recent work indicates that this basic disease resistance is multi-factorial. Here we show that the exudation of root-derived antimicrobial metabolites by Arabidopsis thaliana confers tissue-specific resistance to a wide range of bacterial pathogens. However, a Pseudomonas syringae strain that is both at least partly resistant to these compounds and capable of blocking their synthesis/exudation is able to infect the roots and cause disease. We also show that the ability of this P. syringae strain to block antimicrobial exudation is dependent on the type III secretory system.

Root exudates mediate kin recognition in plants

Though recent work has demonstrated that plants can recognize species, kin versus strangers, and self/non-self roots, no mechanism for identity recognition in plants has yet been found. Here we examined the role of soluble chemicals in signaling among roots. Utilizing Arabidopsis thaliana, we exposed young seedlings to liquid media containing exudates from siblings, strangers (non-siblings), or only their own exudates. In one experiment, root secretions were inhibited by sodium orthovanadate and root length and number of lateral roots were measured. In a second experiment, responses to siblings, strangers, and their own exudates were measured for several accessions (genotypes), and the traits of length of the longest lateral root and hypocotyl length were also measured. The exposure of plants to the root exudates of strangers induced greater lateral root formation than exposure of plants to sibling exudates. Stranger recognition was abolished upon treatment with the secretion inhibitor. In one experiment, plants exposed to sibling or stranger exudates have shorter roots than plants only exposed to their own exudates. This self/non-self recognition response was not affected by the secretion inhibitor. The results demonstrate that that kin recognition and self/non-self are two separate identity recognition systems involving soluble chemicals. Kin recognition requires active secretion by roots.

The rhizobacterial elicitor acetoin induces systemic resistance in Arabidopsis thaliana.

Majority of plant growth promoting rhizobacteria (PGPR) confer plant immunity against a wide range of foliar diseases by activating plant defences that reduce a plant’s susceptibility to pathogen attack. Here we show that Arabidopsis thaliana (Col-0) plants exposed to Bacillus subtilis strain FB17 (hereafter FB17), results in reduced disease severity against Pseudomonas syringae pv. tomato DC3000 (hereafter DC3000) compared to plants without FB17 treatment. Exogenous application of the B. subtilis derived elicitor, acetoin (3-hydroxy-2-butanone), was found to trigger induced systemic resistance (ISR) and protect plants against DC3000 pathogenesis. Moreover, B. subtilis acetoin biosynthetic mutants that emitted reduced levels of acetoin conferred reduced protection to A. thaliana against pathogen infection. Further analysis using FB17 and defense-compromised mutants of A. thaliana indicated that resistance to DC3000 occurs via NPR1 and requires salicylic acid (SA)/ethylene (ET) whereas jasmonic acid (JA) is not essential. This study provides new insight into the role of rhizo-bacterial volatile components as elicitors of defense responses in plants.